Western blot. Protein was run on a 12% SDS page gel and blotted onto PVDF

Transcription

1 Supplemental Data Methods Western blot. Protein was run on a % SDS page gel and blotted onto PVDF membrane (Millipore) by wet transfer. The blots were first incubated with MR monoclonal antibodies () diluted : in blocking buffer containing PBS, % Milk, and.% Tween. Secondary HPR conjugated goat anti-mouse antibody in blocking buffer was applied for hours at room temperature. Following 5 washes in PBS containing.% Tween for 5 minutes per wash, the signal was detected by chemiluminescence. Plasma corticosterone and aldosterone measurement. Blood was collected from mice in heparinized tubes at the end of the L-NAME/Ang-II experiment and plasma was separated for the measurements. Both corticosterone and aldosterone were assayed using Enzyme Immunoassay (EIA) kits (Cayman Chemical) following the manufacture s manuals.

2 Figure legend Figure S. Generation of myeloid specific MR knockout. (A) In mice haboring two conditional MR alleles and LysM-Cre (MRflox/flox;LysM-Cre) the recombined allele (MR-null) is detected by PCR only in isolated macrophages and not in parenchymal tissues. (B) MyMRKO resulted in near complete loss of detectable mrna by qrt-pcr. (C) Western blotting showed near complete loss of MR protein in isolated macrophages. Actin was used as a loading control. MRKO: MyMRKO. Hip: hippocampus; P. Mac: peritoneal macrophage. MW: molecular weight. Figure S. MR regulates multiple functions in macrophages: (A) A heatmap of Affymetrix data demonstrating a broad expression changes in MyMRKO macrophages relative to FC. (B) Gene ontology through GO Miner HTP demonstrates significant biological and cellular functions associated with wound healing were changed by MyMRKO. MRKO: MyMRKO. Figure S3. Responses of resident macrophages to LPS and IL4. (A) M gene expression of resident macrophages isolated from MyMRKO and FC mice and treated with or without LPS. (B) AMφ gene expression of resident macrophages treated with or without LPS. (C) MR gene expression levels in resident macrophages. MRKO: MyMRKO.

3 Figure S4. Overlap of MR and GR mediated effects on macrophage activaiton. Heatmap of Affymetrix data of genes altered > fold by 4 hrs of μm corticosterone treatment to primary peritoneal macrophages from MyMRKO or FC mice. (A) Genes significantly altered by corticosterone. (B) Genes significantly altered by MRKO. (C) GR gene expression in macrophages from FC and MyMRKO mice. MRKO: MyMRKO. Figure S5. Control of macrophage activation by MR and GR. Cluster analysis and heatmap of Affymetrix data illustrating genes coordinately regulated by MR and GR. Three clusters were identified. 3

4 Supplemental Table. Plasma steroid levels in FC and MyMRKO animals with and without L-NAME/Ang-II treatment Control L-NAME/Ang-II FC MyMRKO FC MyMRKO Corticosterone (ng/ml) Aldosterone (pg/ml) 5.48 ± 3.6 (N=5) 8.5 ± 3.53 (N=7).38 ± (N=5) 97.7 ± 3.73 (N=7) 3.34 ± 4.77 (N=5) 9.9 ±.7 (N=7) 7.8 ± (N=5) 34.4 ± 3.96 (N=7) 4

5 References. Gomez-Sanchez, C.E., de Rodriguez, A.F., Romero, D.G., Estess, J., Warden, M.P., Gomez-Sanchez, M.T., and Gomez-Sanchez, E.P. 6. Development of a panel of monoclonal antibodies against the mineralocorticoid receptor. Endocrinology 47:

6 A. B. C. Hip P. Mac MW (kd) FC FC MRKO 5 MR 5 37 Actin Figure S 6

7 A. B. FC MRKO Figure S 7

8 A. TNFα Expression/L IL Expression/L3 5 5 ILβ Expression/L3 4 3 p=. Rantes Expression/L MCP Expression/L Two-way ANOVA: P<. for LPS P=.94 for MRKO P=.98 for interaction P<. for LPS P=.75 for MRKO P=.75 for interaction P<. for LPS P=.38 for MRKO P=.39 for interaction P<. for LPS P=.7 for MRKO P=.75 for interaction P<. for LPS P=.8 for MRKO P=.85 for interaction B. Arg Expression/L Two-way ANOVA: p=.7 p=. * P<. for IL4 P<.5 for MRKO P=.6 for interaction IL Expression/L3 3 P<.5 for IL4 P=.5 for MRKO P<. for interaction YM Expression/L3 4 3 P<. for IL4 P=. for MRKO P=. for interaction Fizz Expression/L3 5 5 P<. for IL4 P=.64 for MRKO P=.63 for interaction Fcrls Expression/L p=.3 * P<. for IL4 P=.55 for MRKO P<.5 for interaction F3a Expression/L p=.4 * p=.35 Ccl7 Expression/L p<. p=.58 Ccl7 Expression/L3 5 5 ILRA Expression/L p=.4 Htra Expression/L C. Two-way P<. for IL4 ANOVA: P=.6 for MRKO P=.8 for interaction MR Expression/L MR Expression/L P<.5 for IL4 P=.5 for MRKO P=.6 for interaction P<.5 for IL4 P=.3 for MRKO P=.64 for interaction P=.96 for IL4 P=.79 for MRKO P=.86 for interaction P=.77 for IL4 P=.4 for MRKO P=.3 for interaction. Two-way P=.7 for LPS ANOVA: P<. for MRKO P=.76 for interaction. P=.9 for IL4 P<. for MRKO P=.5 for interaction Figure S3 8

9 A. B. FC FC+Cort MRKO MRKO+Cort FC FC+Cort MRKO MRKO+Cort C.. GR Expression/8s FC MRKO Figure S4 9

10 FC FC+Cort MRKO MRKO+Cort Figure S5